Advanced chemometric methods for simultaneous quantitation of caffeine, codeine, paracetamol, and p-aminophenol in their quaternary mixture.

Two different multivariate techniques have been applied for the quantitative analysis of caffeine, codeine, paracetamol and p-aminophenol (PAP) in quaternary mixture, namely, Partial Least Squares (PLS-1) and Artificial Neural Networks (ANN). For suitable analysis, a calibration set of 25 mixtures with various ratios of the drugs and PAP impurity were established using a 4-factor 5-level experimental design. The most meaningful wavelengths for the chemometric models were chosen using Genetic Algorithm (GA) as a variable selection technique. By using an independent validation set, the validity of the proposed methods was evaluated. A comparative study was established between the three multivariate models (PLS-1, GA-PLS and GA-ANN). The comparison between the various models revealed that the GA-ANN model was superior at resolving the highly overlapped spectra of this quaternary combination. The drugs were successfully quantified in their pharmaceutical dosage form utilizing the GA-ANN models.

A solvent-free HPLC method for the simultaneous determination of Favipiravir and its hydrolytic degradation product.

During COVID-19 pandemic, Favipiravir (FPV) showed a great efficacy against COVID-19 virus, it produced noticeable improvements in recovery of the patients. The aim of this study was to develop a new, green and simple method for the simultaneous determination of FPV and its acid-induced degradation product (ADP) in its pure and pharmaceutical dosage forms. This method will be key for the inevitable development of FPV solution and inhaler formulations. A green micellar RP-HPLC method was developed using an RP-VDSPHERE PUR 100 column (5 µm, 250 × 4.6 mm) and an isocratic mixed micellar mobile phase composed of 0.02 M Brij-35, 0.1 M SDS and 0.01 M potassium dihydrogen orthophosphate anhydrous and adjusted to pH 3.0 with 1.0 mL min-1 flow rate. The detection was performed at 280 nm with a run time of less than six min. Under the optimized chromatographic conditions, linear relationship has been established between peak area and concentration of FPV and its ADP in the range of 5-100 and 10-100 µg mL-1 with elution time of 3.8 and 5.7 min, respectively. The developed method was validated according to the ICH guidelines and applied successfully for determination of FPV in its pharmaceutical dosage form.

Continuous wavelet transform for solving the problem of minor components in quantitation of pharmaceuticals: a case study on the mixture of ibuprofen and phenylephrine with its degradation products.

The presence of minor components represents a challenging problem in spectrophotometric analysis of pharmaceuticals. If one component has a low absorptivity or present in a low concentration compared to the other components, this will hinder its quantitation by spectrophotometric methods. Continuous Wavelet Transform (CWT) as a signal processing technique was utilized to figure out a solution to such a problem. A comparative study was established between traditional derivative spectrophotometry (Numerical Differentiation, ND) and CWT to indicate the advantages and limitations of each technique and possibility of solving the problem of minor components. A mixture of ibuprofen (IBU) and phenylephrine (PHE) with its degradation products forming a ternary mixture was used for comparing the two techniques. The two techniques were applied on raw spectral data and on ratio spectra data resulting in four methods, namely ND, CWT, Derivative Ratio-Zero Crossing (DRZC) and Continuous Wavelet Transform Ratio-Zero Crossing (CWTR-ZC) methods. By comparing the results in laboratory prepared mixtures, CWT technique showed advantages in analysis of mixtures with minor components than ND. The proposed methods were validated according to the ICH guideline Q2(R1), where their linearity was established with correlation coefficient ranging from 0.9995 to 0.9999. The linearity was in the range 3-40 µg/mL for PHE in all methods, while for IBU it was 20-180 and 30-180 µg/mL in CWT and ND methods, respectively. The CWT methods were applied for quantitative determination of the drugs in their dosage form showing the ability of the methods to quantitate minor components in pharmaceutical formulations.

Development of an eco-friendly HPLC method for the stability indicating assay of binary mixture of ibuprofen and phenylephrine.

The development and validation of the stability indicating HPLC technique has contributed to the understanding of the stability profile of ibuprofen (IBU) and phenylephrine (PHE). Stability profile was achieved for PHE; the drug was found to be liable to be influenced by stress oxidative conditions; two oxidative degradants (Deg1 & Deg2) were formed and their structures were confirmed using IR and mass spectrometry. The drugs and degradation products were successfully separated using a gradient elution method on YMC-C8 column with 0.1% hexanesulfonic acid and acetonitrile as a mobile phase at pH 6.6. The flow rate was 1.0 mL/min, and a diode array detector operating at 220 nm was used for UV detection. The retention times of degradants Deg1, Deg2, ibuprofen (IBU), and phenylephrine hydrochloride (PHE) were 2.0, 2.2, 3.2 and 7.0 min, respectively. The proposed method was validated with respect to linearity, accuracy, precision, specificity, and robustness using ICH guidelines. The linearities of ibuprofen and phenylephrine hydrochloride were in the range of 10-100 µg/mL and 0.3-10 µg/mL, respectively. The % recoveries of the two drugs were found to be 100.75 ± 1.44%, 99.67% ± 1.67, and the LOD was found to be 2.75/mL and 0.09/mL for IBU, and PHE, respectively. The method was successfully applied to the estimation of ibuprofen and phenylephrine hydrochloride combination in pharmaceutical dosage form. The proposed technique was validated using ICH guidelines and its greenness was assessed according to Analytical Eco Scale metric (AES). Molecular docking was used to assess the two drugs and PHE oxidative degradants interaction with the stationary phase and to confirm the outcomes of the proposed method with regard to the order of elution of the two drugs and PHE degradation products. Eco-friendly and environmental safety were assessed through the application of one of the most applicable greenness assessment tool; Analytical Eco Scale metric (AES).

Cytotoxic Activity of Phytoconstituents Isolated from Monotheca buxifolia against Hepatocellular Carcinoma Cell Line HepG2: In Vitro and Molecular Docking Studies.

Natural products and conventional chemotherapeutic drugs are believed to enhance anticancer treatment efficacy while lowering toxicity. The current study investigates the cytotoxic and apoptogenic effects of Monotheca buxifolia bioactive compounds on HepG2 cell lines. MTT assay was used to assess the effect on the viability of HepG2 cells. Morphological changes were investigated. Annexin-V-FITC/PI was used to demonstrate apoptotic activity. A molecular dynamics simulation study was carried out to investigate the compound binding pattern in the active site of the PPRAδ protein. MTT and annexin V-FITC/PI assays revealed that the isolated compounds lauric acid, oleanolic acid, and bis(2-ethylhexyl) phthalate inhibited the growth of hepatocellular cancer cells. The IC50 value for lauric acid was 56.46 ± 1.20 µg/mL, 31.94 ± 1.03 µg/mL for oleanolic acid, and 83.80 ± 2.18 µg/mL for bis(2-ethylhexyl) phthalate. Apoptosis was observed in 29.5, 52.1 and 22.4% of HepG2 cells treated with lauric acid, oleanolic acid, and bis(2-ethylhexyl) phthalate, respectively, after 24 h of treatment. Morphological assays and Hoechst staining microscopy revealed that the treatment caused morphological changes in the cell membrane and nuclear condensation. The high fluctuation indicates that various interactions were highly potent and widely adopted, and vice versa. Oleanolic acid displayed high residue fluctuation, remaining stable in the active site of the PPRAδ protein and involved in various interactions while remaining locally fluctuating in the binding sites of the other two compounds. These findings concluded that lauric acid, oleanolic acid, and bis(2-ethylhexyl) phthalate have a significant apoptogenic effect against HepG2 cells in inducing apoptosis. Our findings suggest that these bioactive compounds could be used as adjuvant therapies.

Exploring the Therapeutic Properties of Alga-Based Silver Nanoparticles: Anticancer, Antibacterial, and Free Radical Scavenging Capabilities.

The current study was designed to evaluate the antioxidant, anticancer and antimicrobial activities of silver nanoparticles (AgNPs) biosynthesized by Spirulina platensis extract. The biosynthesized silver nanoparticles were characterized using Fourier transform infrared (FT-IR) analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD) analysis. The antioxidant activity of the biosynthesized AgNPs were determined via DPPH radical scavenging assay while its anticancer activity was determined using the MTT assay. The antimicrobial activity of the biosynthesized AgNPs were analyzed by disc diffusion method. Spirulina platensis acts as a reducing and capping agent. The efficacy of silver nanoparticles (AgNPs) in inhibiting the growth of Gram-negative bacteria, specifically Acetobacter, Klebsiella, Proteus vulgaris, and Pseudomonas aeruginosa, was assessed by the utilisation of the diffusion method. The study aimed to evaluate the efficacy of biosynthesized silver nanoparticles (AgNPs) against many strains of Pseudomonas aeruginosa bacteria. The findings of the study revealed that when administered in doses of 50 µl, 75 µl, and 100 µl, the largest observed zone of inhibition corresponded to measurements of 10.5 mm, 14 mm, and 16 mm, respectively. A zone of inhibition with dimensions of 8 mm, 10.5 mm, and 12 mm was detected during testing against Acetobacter at concentrations of 50 µl, 75 µl, and 100 µl, respectively. The findings also indicate that there is a positive correlation between the concentration of AgNP and the DPPH scavenging ability of silver nanoparticles. The percentage of inhibition observed at concentrations of 500 µg/ml, 400 µg/ml, 300 µg/ml, 200 µg/ml, and 100 µg/ml were recorded as 80±1.98, 61±1.98, 52±1.5, 42±1.99, and 36±1.97, respectively. In addition, it was observed that the silver nanoparticles exhibited the greatest antioxidant activity at a concentration of 500 g/ml, with a measured value of 80.89±1.99. The IC-50 values, representing the inhibitory concentration required to achieve 50 % inhibition, were found to be 8.16, 19.15, 30.14, 41.13, and 63.11 at inhibition levels of 36±1.97, 42±1.99, 52±1.5, 61±1.98, and 80±1.98, respectively.

Fluorescence imaging approaches for eco-friendly determination of perampanel in human plasma and application for therapeutic drug monitoring.

Antiepileptic drugs are among the most common medications that require therapeutic drug monitoring (TDM). Indeed, TDM provides a realistic approach to adjust drug doses for epilepsy based on plasma concentrations to optimize its clinical outcome. The most common technique for TDM is high-performance liquid chromatography, which has a very low green profile among analytical techniques. Perampanel (PER) is an inherently fluorescent compound that its fluorophore readily allows sensitive and quantitative measurements. This paper describes the development and validation of a sensitive, specific, and eco-friendly spectrofluorimetric method for the determination of PER. Experimental parameters affecting fluorescence intensity of the compound, including solvent dilution, temperature, and excitation wavelength, were studied and optimized. The developed spectrofluorimetric method was established in acetonitrile at λex  = 295 nm and λem  = 431 nm over a concentration range of 5-60 ng/ml. The adopted method was applied for the determination of PER in human plasma; it was effective in the range of 15-50 ng/ml. The proposed method was found to be sensitive and specific for PER and can be applied successfully in TDM of PER and in quality control laboratories.

Thiourea derivatives inhibit key diabetes-associated enzymes and advanced glycation end-product formation as a treatment for diabetes mellitus.

This study was designed to screen novel thiourea derivatives against different enzymes, such as α-amylase, α-glucosidase, protein tyrosine phosphatase 1 B, and advanced glycated end product (AGEs). A cytotoxicity analysis was performed using rat L6 myotubes and molecular docking analysis was performed to map the binding interactions between the active compounds and α-amylase and α-glucosidase. The data revealed the potency of five compounds, including E (1-(2,4-difluorophenyl)-3-(3,4-dimethyl phenyl) thiourea), AG (1-(2-methoxy-5-(trifluoromethyl) phenyl)-3-(3-methoxy phenyl) thiourea), AF (1-(2,4-dichlorophenyl)-3-(4-ethylphenyl) thiourea), AD (1-(2,4-dichlorophenyl)-3-(4-ethylphenyl) thiourea), and AH (1-(2,4-difluorophenyl)-3-(2-iodophenyl) thiourea), showed activity against α-amylase. The corresponding percentage inhibitions were found to be 85 ± 1.9, 82 ± 0.7, 75 ± 1.2, 72 ± 0.4, and 65 ± 1.1%, respectively. These compounds were then screened using in vitro assays. Among them, AH showed the highest activity against α-glucosidase, AGEs, and PTP1B, with percentage inhibitions of 86 ± 0.4% (IC50  = 47.9 µM), 85 ± 0.7% (IC50  = 49.51 µM), and 85 ± 0.5% (IC50  = 79.74 µM), respectively. Compound AH showed an increased glucose uptake at a concentration of 100 µM. Finally, an in vivo study was conducted using a streptozotocin-induced diabetic mouse model and PTP1B expression was assessed using real-time PCR. Additionally, we examined the hypoglycemic effect of compound AH in diabetic rats compared to the standard drug glibenclamide.

Predictive Factors for Magnetic Resonance Imaging Changes Suggestive of Demyelination in Adult Patients with Uveitis Scanned Prior to Commencing Adalimumab Therapy.

PURPOSE: To report the predictive clinical factors for abnormal magnetic resonance imaging (MRI) scans suggestive of demyelination by analysis of MRI's performed for adult non-infectious uveitic patients prior to commencing adalimumab therapy. METHODS: Retrospective case review of 240 patients was conducted in a single tertiary institution between November 2017 and March 2020. Aetiology of underlying disease, clinical characteristics, and MRI outcomes were analysed. RESULTS: The presence of bilateral idiopathic intermediate uveitis (IIU) (p = .0048) and neurological symptoms (p = .028) were highly predictive of an abnormal MRI strongly suggestive of demyelination (MRSSD); 5 out of 64 scans (7.8%) with these clinical characteristics had MRSSD. CONCLUSIONS: Tumor necrosis factor antagonist-induced demyelination is a concern in adalimumab use. We propose an MRI screening protocol to identify those at high risk of demyelination; positive results can be maximised by screening all patients with IIU and those with neurological symptoms.

Dietary supplement mislabelling: case study on selected slimming products by developing a green isocratic HPLC method for their quality control.

Nowadays, a huge population consumes Dietary supplements for losing weight. Products are often claimed as botanical blends, yet they aren't necessarily safe. Misleading labels are also very common. Thus, validated analytical methods for a wide range of slimming compounds are highly needed. Herein, we present a simple HPLC/PDA method for the quantitation of seven popular slimming ingredients. Studied compounds were Caffeine, Raspberry Ketone, trans-Resveratrol, p-Synephrine, p-Octopamine, p-Hordenine and 2-phenethylamine. After optimization, separation was carried out on a C18 column and mobile phase was a mixture of Acetonitrile:Water containing 0.1% phosphoric acid (50:50, %v/v). The last compound was eluted at 9.76 min. Separation was efficient showing baseline- separated symmetric peaks, without using any gradient programs, organic mobile phase modifiers or modified stationary phases. Method validation was done following ICH guidelines. Calibration curves were linear over wide concentration ranges and calculated LOD values were in the range 0.02-0.09 µg/mL. Method greenness was assessed using Analytical Eco-scale, GAPI and AGREE metric tools. Further, four random sample products purchased from online supplement stores were assayed. Results proved some mislabeling actions. To support our findings, standard addition was carried out and average % recoveries were 96.67 - 101.44% with standard deviation ≤ 2.83 between measurements.

Cisplatin and oleanolic acid Co-loaded pH-sensitive CaCO3 nanoparticles for synergistic chemotherapy.

Despite being one of the most potent anticancer agents, cisplatin (CDDP) clinical usage is limited owing to the acquired resistance and severe adverse effects including nephrotoxicity. The current work has offered a unique approach by designing a pH-sensitive calcium carbonate drug delivery system for CDDP and oleanolic acid (OA) co-delivery, with an enhanced tumor efficacy and reduced unwanted effects. Micro emulsion method was employed to generate calcium carbonate cores (CDDP encapsulated) followed by lipid coating along with the OA loading resulting in the generation of lipid-coated cisplatin/oleanolic acid calcium carbonate nanoparticles (CDDP/OA-LCC NPs). In vitro biological assays confirmed the synergistic apoptotic effect of CDDP and OA against HepG2 cells. It was further verified in vivo through the tumor-bearing nude mice model where NPs exhibited enhanced satisfactory antitumor efficacy in contrast to free drug solutions. In vivo pharmacokinetic study demonstrated that a remarkable long circulation time with a constant therapeutic concentration for both drugs could be achieved via this drug delivery system. In addition, the in vivo imaging study revealed that DiR-loaded NPs were concentrated more in tumors for a longer period of time as compared to other peritoneal tissues in tumor bearing mice, demonstrating the site specificity of the delivery system. On the other hand, hematoxylin and eosin (H&E) staining of Kunming mice kidney tissue sections revealed that OA greatly reduced CDDP induced nephrotoxicity in the formulation. Overall, these results confirmed that our pH-sensitive dual loaded drug delivery system offers a handy direction for effective and safer combination chemotherapy.

Anatomical, histochemical and surface ultrastructural characteristics of cavum nasi of the common quails (Coturnix coturnix. Erlangeri).

Ten normal, mature and common quails were used to study in detail the gross anatomy, histochemical and surface ultrastructural characteristics of the nasal cavity. The relationship between the structure and function of the nasal cavity also were assessed. The quail nasal cavity was divided into the vestibule, nasal cavity proper and fundus. The nasal cavity began rostrally by two slit-like external nares located laterally in the middle third of the upper beak. A previous authors stated that no rostral concha but the current study record that the rostral nasal concha was located opposite the nostrils and exhibited a C-shaped appearance in transverse section and was 5 mm long and 3 mm wide. The middle nasal concha was narrow and elongated. The caudal nasal concha was spherical, located caudodorsal to the rostral nasal concha and measured 2 mm in diameter. The infraorbital sinus was a roughly triangular cavity situated immediately rostral to the orbit. The histological and surface ultrastructural study of the nasal cavity of common quail did not studied previously. Histologically, the cavum nasi was composed of three regions: vestibule, respiratory and olfactory. The vestibule was lined with stratified squamous epithelium that was keratinized rostrally and non-keratinized caudally. The respiratory region was covered by pseudostratified columnar epithelium. Intra-epithelial mucous glands were present in the respiratory region and displayed a strong reaction with Alcian blue. The lining epithelium in the olfactory region was pseudostratified and contained olfactory, supporting and basal cells.

Effect of the Prenatal Exposure of Khat on the Skeleton of Developing Rabbit Embryo: Morphometric and Gross Anatomical Study.

Background: Khat leaves are chewed by many people worldwide, mainly in Africa and the southwest Arabian peninsula. Materials and methods: 27 apparently healthy adult New Zealand rabbits (3 males & 24 females) of 2.5 ± 0.5 kg body weight were used in this work. The animals were divided into 4 groups (control, low, medium and high doses groups). The khat extract was administered orally during (8th -18th) day of gestation. The mothers were sacrificed on the 29th day of gestation. The skeletons of the fetuses were stained with Alizarin Red S and Alcian Blue. Results: Skeletal examination of the fetuses of treated dams showed several types of malformations and variations in all treated groups such as unossified phalanges, deformed sternum, completely unossified vertebral lamina and opened anterior and posterior fontanelles, and reduced length of limb long bones. Conclusion: These findings support the teratogenic effect of the khat on the developing rabbit fetus.

Advanced chemometric methods as powerful tools for impurity profiling of drug substances and drug products: Application on bisoprolol and perindopril binary mixture.

Impurity profiling has a rising importance nowadays due to the increased health problems associated with impurities and degradation products found in several drug substances and formulations. Three advanced, accurate and precise chemometric methods were developed as impurity profiling methods for a mixture of bisoprolol fumarate (BIS) and perindopril arginine (PER) with their degradation products which represent drug impurity or a precursor to such impurity. The methods applied were Partial Least Squares (PLS-1), Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) and Artificial Neural Networks (ANN). Genetic Algorithm (GA) was used as a variable selection tool to select the most significant wavelengths for the three chemometric models. For proper analysis, a 5-factor 5-level experimental design was used to establish a calibration set of 25 mixtures containing different ratios of the drugs and their degradation products (impurities). The validity of the proposed methods was assessed using an independent validation set. The designed models were able to predict the concentrations of the drugs and the degradation products/impurities in the validation set and pharmaceutical formulation. The proposed methods presented a powerful alternative to traditional and expensive chromatographic methods as impurity profiling tools.

Identification of a Potential Vaccine against Treponema pallidum Using Subtractive Proteomics and Reverse-Vaccinology Approaches.

Syphilis, a sexually transmitted infection, is a deadly disease caused by Treponema pallidum. It is a Gram-negative spirochete that can infect nearly every organ of the human body. It can be transmitted both sexually and perinatally. Since syphilis is the second most fatal sexually transmitted disease after AIDS, an efficient vaccine candidate is needed to establish long-term protection against infections by T. pallidum. This study used reverse-vaccinology-based immunoinformatic pathway subtractive proteomics to find the best antigenic proteins for multi-epitope vaccine production. Six essential virulent and antigenic proteins were identified, including the membrane lipoprotein TpN32 (UniProt ID: O07950), DNA translocase FtsK (UniProt ID: O83964), Protein Soj homolog (UniProt ID: O83296), site-determining protein (UniProt ID: F7IVD2), ABC transporter, ATP-binding protein (UniProt ID: O83930), and Sugar ABC superfamily ATP-binding cassette transporter, ABC protein (UniProt ID: O83782). We found that the multiepitope subunit vaccine consisting of 4 CTL, 4 HTL, and 11 B-cell epitopes mixed with the adjuvant TLR-2 agonist ESAT6 has potent antigenic characteristics and does not induce an allergic response. Before being docked at Toll-like receptors 2 and 4, the developed vaccine was modeled, improved, and validated. Docking studies revealed significant binding interactions, whereas molecular dynamics simulations demonstrated its stability. Furthermore, the immune system simulation indicated significant and long-lasting immunological responses. The vaccine was then reverse-transcribed into a DNA sequence and cloned into the pET28a (+) vector to validate translational activity as well as the microbial production process. The vaccine developed in this study requires further scientific consensus before it can be used against T. pallidum to confirm its safety and efficacy.

Using mathematical models to evaluate germination rate and seedlings length of chickpea seed (Cicer arietinum L.) to osmotic stress at cardinal temperatures.

Cicer arietinum is the 3rd most important cool season legume crop growing in vast arid and semi-arid regions of the world. A lab experiment was designed using hydrothermal time model (HTT) to investigate the chickpea seed germination (SG) behavior, cardinal temperatures and germination responses across fluctuating temperatures (Ts) and water potentials (Ψs). Seeds of chickpea var. NIFA 1995 were germinated at six constant Ts (7, 14, 21, 28, 35 and 42°C) each having the following five water potentials: 0, -0.2, -0.4-0.6 and -0.8 MPa. Germination percentage (G%) decreased significantly at (*P ≤ 0.05) from 86.7% at 28°C in -0.2 MPa to 10% in -0.2 MPa at 7°C. The germination rate (GR = 1/t50) against different T percentiles exhibited that linear increase was observed in the GR pattern above and below the To. Based on the confidence intervals of the model coefficients and (R2: 0.96), the average cardinal temperatures were 4.7, 23 and 44.2°C for the base (Tb), optimal (To) and ceiling (Tc) temperatures respectively. θT1 value was observed maximum at 28°C in -0.2 MPa and decreases with decreasing Ψ (-0.8 MPa). In comparison with control, the θT2 value was also highest in -0.2 MPa at 28°C. The thermal time (TT) concept is well fitted to germination fraction data in distilled water with an R2 value increasing 0.972. The hydro time constant (θH) increased with an increase in T to To and then decreased when T>To. The ѱb(50) irregularly varied with increasing T, σΨb was also recorded lowest (0.166 MPa) at 28°C and highest (0.457 MPa) at 7°C. Based on the statistical analysis, cardinal temperatures, hydrothermal time constant (θHTT) and germination findings the HTT gives an insight into the interactive effect of T and Ψ on seed germination time courses under varying environmental conditions.

Heavy metals bioaccumulation and subsequent multiple biomarkers based appraisal of toxicity in the critically endangered Tor putitora.

The construction of hydropower projects discharges effluents to aquatic bodies. The effluents consist of different chemicals including heavy metals. The current study assessed the effects of effluents discharged from an under-construction hydropower project on the bioaccumulation of heavy metals in the tissues of critically endangered Tor putitora (Hamilton, 1822) in the river Panjkora. The subsequent toxic impacts of higher bioaccumulation of heavy metals on different biochemical, hematological, and serum biochemical profiles were also studied. Different biochemical changes were observed in the tissues of T. putitora including stress biomarkers such as reactive oxygen species, lipid peroxidation, total protein contents, antioxidant enzymes (peroxidase, superoxide dismutase, catalase, reduced glutathione, glutathione reductase, and glutathione-s-transferase), acetylcholinesterase, and whole-body cortisol. The hematotoxic effects were also observed as the count of red blood cells, hematocrit, and hemoglobin decreased whereas the count of white blood cells increased. Serum biochemical analysis revealed that cholesterol, urea, total bilirubin, and glucose concentration increased, whereas total proteins and albumin decreased with an increase in the concentration of heavy metals across the sampling sites. The fish from the river was found to be under severe stress as compared to the fish from the reference site. To mitigate the current scenario, stocking fish in an appropriate amount is suggested. The fish diversity and water quality should be assessed at regular intervals to avoid further deterioration and diversity loss. The safety and conservation of wild fisheries should be ensured by implementing strict environmental protection and fishing laws.

Phylogenetic analysis of the 5' untranslated region of HCV from cirrhotic patients in Khyber Pakhtunkhwa, Pakistan.

Hepatitis C virus (HCV), a small, single-stranded RNA virus with a 9.6 kb genome, is one of the most common causes of liver diseases. Sequencing of the 5' untranslated region (UTR) is usually used for HCV genotyping, but it is less important in numerous subtypes due to its scarce sequence variations. This study aimed to identify genotypes using the 5' UTR of HCV from cirrhotic patients of Khyber Pakhtunkhwa (KP). Serum RNA samples (44) were screened by real time PCR to determine the HCV viral load. Nested PCR was performed to identify cDNA and the 5' UTR. The HCV 5' UTR was sequenced using the Sanger method. MEGA-7 software was used to analyze evolutionary relatedness. After 5' UTR sequencing, 26 samples (59%) were identified as genotype 3, and 2 samples (6%) were identified as genotypes 1, 2 and 4. The most predominant genotype was 3a, and genotype 4 was rarely reported in the phylogenetic tree. Analysis of the HCV 5' UTR is an efficient alternative method for confirmation of various genotypes. Phylogenetic analysis showed that genotype 3 was dominant in the area of KP, Pakistan.

Quality control of dietary supplements: An economic green spectrofluorimetric assay of Raspberry ketone and its application to weight variation testing.

Although Dietary supplements are readily accessible and extensively used worldwide, they are inadequately regulated and consumers are victims of manufacturers' fraud. Thus, quality regulations are required to ensure safety of products available to the public. We propose the first native spectrofluorimetric quality control assay of raspberry ketone, a popular dietary supplement ingredient for weight loss. This work relies on the constant wavelength synchronous scan of the Raspberry Ketone native fluorescence, overcoming the demerits of conventional excitation/ emission spectra. For the best measurement conditions, several parameters were optimized including Δλ value, diluting solvent, medium pH and the effect of surfactants/ macromolecules. In aqueous medium (Δλ = 110 nm), a linear relationship exists between synchronous fluorescence intensity at peak maximum 405.6 nm and solution concentration in the range 300-1500 ng/mL. Method sensitivity was recorded with LOD and LOQ values 60.63 and 183.72 ng/mL; respectively. Validation was done in accordance to International Conference on Harmonization (ICH) guidelines. This simple procedure was successfully applied to the analysis of Raspberry Ketone in commercially available dietary supplement capsules with average recovery 98.67% ± 1.74 and further extended to weight variation testing following the official United States Pharmacopeial (USP) guidelines. Finally, green assessment was done using the ''Analytical Eco-scale'' tool. The total score was 89/100 points revealing excellent greenness of our proposal. Our proposal is simple, eco-friendly and cheap. It can be conveniently adopted for routine quality control practices especially in developing countries.